Organ‐transplant recipients (OTRs) on a new class of immunosuppressants, rapamycin and its analogues, have reduced cutaneous Squamous Cell Carcinomas (cSCCs). Rapamycin, an mTORC1 inhibitor, is also a known autophagy inducer in experimental models. Autophagy, which literally means self‐eating, is a cell survival mechanism but can also lead to cell death. Therefore, the main hypothesis behind this work is that rapamycin prevents epidermal tumourigenesis by either affecting epidermal mTOR regulation of autophagy and/or selectively affecting epidermal AKT isoform activity. Epidermal keratinocytes move from the proliferating basal layer upwards to the granular layers where they terminally differentiate, forming a layer of flattened, anucleate cells or squames of the cornified layer which provides an essential environmental barrier. However, epidermal terminal differentiation, a specialised form of cell death involving organelle degradation, is poorly understood. The work presented in this thesis shows that analysis of the autophagy marker expression profile during foetal epidermal development, indicates autophagy is constitutively active in the terminally differentiating granular layer of epidermis. Therefore, I hypothesize that autophagy is a mechanism of organelle degradation during terminal differentiation of granular layer keratinocytes. In monolayer keratinocytes, activation of terminal differentiation is accompanied by autophagic degradation of nuclear material, nucleophagy. This suggests that constitutive autophagy is a pro‐death mechanism required for terminal differentiation. In cultured keratinocytes and in epidermal cultures, rapamycinmediated mTORC1 inhibition strongly increases AKT1 activity as well as up‐regulates constitutive granular layer autophagy promoting terminal differentiation. Therefore, autophagy is an important fundamental process in keratinocytes which may be the mechanism by which terminally differentiating keratinocytes of the epidermal granular layer degrade their organelles required for barrier formation. This may have implications for the treatment of patients with barrier defects like psoriasis. In immunosuppressed OTRs, rapamycin may promote epidermal autophagy and AKT1 activity adding to its anti‐tumourigenic properties.

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Το σηματοδοτικό μονοπάτι Hippo παίζει καθοριστικό ρόλο στην ομοιόσταση ιστών και οργάνων κατά την εμβρυογένεση και την ενήλικο ζωή ενώ όλο και περισσότερες μελέτες αναδεικνύουν τη σημασία του στην καρκινογένεση. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της έκφρασης των πρωτεϊνών YAP, TAZ, Ε-cadherin και b-catenin, στο Βασικοκυτταρικό καρκίνωμα του δέρματος (ΒΚΚ) και η συσχέτιση των ευρημάτων με την ενεργοποίηση της σηματοδοτικής οδού HIPPO, με την αλληλεπίδραση των σηματοδοτικών μονοπατιών Wnt/b-catenin και Hippo/YAP και με κλινικοπαθολογοανατομικές παραμέτρους του νεοπλάσματος. . Μέθοδοι και αποτελέσματα: Με την τεχνική της ανοσοϊστοχημείας ελεγχθηκε η έκφραση των πρωτεϊνών ΥΑΡ, ΤΑΖ, Ε-cadherin και b-catenin σε τομές παραφίνης μονιμομοποιημένου σε φορμόλη ιστού 95 περιστατικών ανθρώπινου ΒΚΚ. Η έκφραση και των δύο βασικών επιτελεστικών μορίων του Hippo (YAP,TAZ) βρέθηκε αυξημένη στον πυρήνα όλων των ιστολογικών τύπων ΒΚΚ και ήταν σημαντικά αυξημένη στους υψηλού κινδύνου υπότυπους. Τα μικροοζώδη, διαφοροποιούνταν τόσο από τα διηθητικά όσο και από τους χαμηλού κινδύνου υπότυπους εμφανίζοντας ενδιάμεση έκφραση για την ΥΑΡ όμως την υψηλότερη έκφραση για την ΤΑΖ μεταξύ όλων των ιστολογικών υποτύπων. Η E-cadherin στα περιστατικά μας διατηρούσε σε μεγάλο βαθμό τη μεμβρανική της έκφραση αν και μειωμένη σε σχέση με τη φυσιολογική επιδερμίδα, χωρίς σημαντικές διαφορές μεταξύ των ιστολογικών τύπων ή μεταξύ του όγκου συνολικά και της διηθητικής παρυφής. Τέλος, στα μισά περίπου περιστατικά μας διαπιστώθηκε πυρηνική έκφραση της β-catenin η οποία ήταν σημαντική στα διηθητικά σε σχέση με τα οζώδη, ενώ επίσης τα διηθητικά ΒΚΚ εμφάνιζαν τα υψηλότερα ποσοστά κυτταροπλασματικής έκφρασης της β-catenin μεταξύ όλων των ιστολογικών τύπων. Επίσης, η μεμβρανική έκφραση της β-catenin στο διηθητικό μέτωπο των υψηλού κινδύνου υποτύπων ήταν μειωμένη σε σχέση με τους χαμηλού κινδύνου. Συμπεράσματα: Το μονοπάτι Hippo φαίνεται ότι συμβάλλει στην παθογένεια του ΒΚΚ με έμφαση στους υψηλού κινδύνου υποτύπους, έχοντας κατά κύριο λόγο ογκογόνο δράση. Στην περίπτωση του ΒΚΚ, η Ε-cadherin δεν φαίνεται να αποτελεί ανωφερή ρυθμιστή του Hippo καθώς η διατήρηση της μεμβρανικής της έκφρασης δεν συνοδεύεται από σημαντικές αλλαγές στην υποκυτταρική εντόπιση της ΥΑΡ, γεγονός που ίσως αντανακλά τον ιστό προέλευσης του όγκου και οι νεοπλασματικές φωλεές διατηρούν αξιοσημείωτη κυτταρική συνοχή και επιθηλιακό φαινότυπο ακόμη και στην περίπτωση των επιθετικών υποτύπων. Η προφανής απαίτηση για Wnt/β-catenin σηματοδότηση στα ΒΚΚ θα πρέπει να ερευνηθεί περαιτέρω καθώς ενδέχεται να βρίσκεται υπό αρνητική ρύθμιση από παράγοντες όπως η ενεργοποίηση της non canonical Wnt σηματοδοτικής οδού που δρά ανταγωνιστικά ως προς την canonical Wnt σηματοδοτική οδό και η έκφραση σε υψηλά επίπεδα της E-cadherin στις μεμβράνες. Η παρατηρούμενη βιβλιογραφική ετερογένεια ως προς τη σημασία της Wnt σηματοδότησης στην παθογένεια του ΒΚΚ πιθανόν να αντανακλά βιολογική ετερογένεια ως προς την απαίτηση για Wnt σηματοδότηση εν γένει στο ΒΚΚ που πιθανόν αντικατοπτρίζει διαφορετική συμβολή της Wnt σηματοδοτικής οδού στα διάφορα στάδια εξέλιξης του όγκου ή/και στους διαφορετικούς ιστολογικούς υπότυπους. / Hippo pathway has emerged as a crucial component of organ and tissue homeostasis. Increasing number of studies highlight it’s significance in carcinogenesis. The aim of this study was to determine the expression of YAP, TAZ, Ε-cadherin and b-catenin in human Basal Cell Carcinoma (BCC) and correlate it with the activation status of Hippo signaling pathway, the interaction of Wnt/b-catenin και Hippo/YAP signaling pathways and clinicopathological features of the tumor. Materials and results: Paraffin-embedded tissue sections from 95 human BCC cases were processed by immunohistochemistry for the expression of YAP, TAZ, Ε-cadherin and b-catenin. Nuclear expression of both Hippo effector proteins (YAP,TAZ) was observed in 100% of cases and was strongly corellated with the high risk subtypes. Micronodular differed from both the invasive and low risk subtypes by showing intermediate nuclear YAP expression and the highest nuclear TAZ expression among all BCC subtypes tested. E-cadherin expression in our cases was largely membranous, though reduced compared to normal epidermis, with no significant differences between histologic subtypes or between the tumor overall and it’s invasive front. Finaly, nuclear β-catenin expression was observed in about half of our cases and was significantly increased in the infiltrative compared to the nodular subtype with the infiltrative BCC’s having the highest cytoplasmic β-catenin expression among all subtypes tested. Moreover, membranous β-catenin expression at the invasive front was significantly reduced in the high risk subtypes when compared to the low risk. Conclusions: We believe that Hippo signaling pathway holds a critical oncogenic role in the pathogenesis of BCC, expecially in the high risk subtypes. In the case of BCC, Ε-cadherin doesn’t appear to act as an upstream regulator of Hippo because it’s relatively stable membranous expression doesn’t seem to follow any variations in the subcellular localization of YAP, an observation probably reflecting the fact that skin is the tissue of origin for BCC. Neoplastic nests remain remarkably cohesive and retain an epithelial phenotype even in the high risk variants. The obvious demand for Wnt/β-catenin signaling in BCC development needs to be further invastigated because it seems likely being under negative regulation by factors like activation of the non canonical Wnt signaling cascade and increased expression levels of membranous E-cadherin. The observed variability in the literature regarding the significance of canonical Wnt signaling in BCC pathogenesis could be reflecting biological variability in the contribution of Wnt signaling in BCC pathogenesis overall, corresponding to differencies in Wnt signaling between the various stages of neoplastic development or/and the different histologic subtypes .

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Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2014-12-19T16:34:27Z No. of bitstreams: 0 / Made available in DSpace on 2014-12-19T16:34:27Z (GMT). No. of bitstreams: 0 / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Os microRNAs (miRNAs, miRs) são pequenos RNAs não codificadores presentes em diferentes organismos. Esses RNAs regulam a tradução de genes alvos por meio de ligação seqüência-específica a RNAs mensageiros (mRNAs). Dependendo do grau de complementaridade, podem inibir a tradução e/ou induzir a degradação desses mRNAs. No presente estudo, foi investigado por PCR em tempo real o padrão de expressão de quatro microRNAs (miR-21, -205, -342 e let-7a ) em quatro linhagens celulares derivadas de tumores da cavidade oral e da faringe (FaDu, Hep-2, SCC9 e UM-SCC-38), em queratinócitos orais normais e em amostras de tumor e margens cirúrgicas pareadas de 34 pacientes com carcinomas epidermóides de cabeça e pescoço (CECP). Foi também investigada a correlação da expressão dos MiRs de interesse com as características clinicopatológicas de pacientes com CECP. Nas linhagens celulares, os níveis dos miRs foram similares ou mais baixos que os de queratinócitos normais, ou os miRs não se expressaram. Somente o miR-342 mostrou níveis elevados na linhagem FaDu. Em células Hep-2 tratadas com estradiol, a expressão de miR-let-7a mostrou-se reduzida. Em tumores primários, níveis baixos de miR-let-7a foram observados em carcinomas de soalho de boca e laringe. A expressão de miR-21, -205 e -342 mostrou grande variabilidade entre as amostras e foi reduzida em um dos sítios anatômicos. Não foi observada correlação entre a expressão dos miRs e as características clinicopatológicas dos pacientes com CECP. A análise de três genes alvo candidatos (LYZ, MGLL e SPRR3) mostrou, em carcinomas de soalho de boca e laringe, associação positiva entre a expressão de miR-let-7a e de seu alvo predito MGLL, uma lipase que pode favorecer o fenótipo maligno aumentando os níveis de ácidos graxos livres e sinais lipídicos oncogênicos. O significado dessa associação não pode ser deduzida dos experimentos realizados pelo presente trabalho. / MicroRNAs (miRNAs, miRs) are small, non-coding RNAs present in different organisms. They regulate the translation of target genes through sequence specific binding to mRNA. Depending on the degree of sequence complimentary, they can inhibit translation and/or degradation of target mRNAs In the present study, we used real time PCR to investigate the expression pattern of four microRNAs (miR-21, -205, -342 e let-7a ) in four cell lines derived from tumors of oral cavity and pharinx (FaDu, Hep-2, SCC9 e UM-SCC-38), in normal oral keratinocytes and in matched tumor / surgical margin samples from 34 patients with head and neck squamous cell carcinomas (HNSCC). We also aimed to correlate the miR expression with the clinicopathological features in HNSCC. In cell lines, the miR levels were similar or lower than those in normal keratinocytes, or even absent. Only miR-342 showed high levels in FaDu cell line. In Hep-2 cells treated with estradiol, miR-let-7a expression was reduced. In primary tumors, low miR-let-7a levels were observed in floor of the mouth and larynx carcinomas. The expression of miR-21, -205 and -342 showed high variability between samples and was reduced in one anatomical site. No correlation was observed between miR expression and clinopathological features of head and neck cancer patients. The analysis of three potential target genes (LYZ, MGLL e SPRR3) showed, in floor of the mouth and larynx carcinomas, a positive correlation between the expression of miR-let-7a and its predicted target gene MGLL, a lipase that may support the malignant phenotype by increasing levels of free fatty acids and oncogenic lipid signals. The meaning of such association was not clear from our data.

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Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2014-12-19T16:34:27Z No. of bitstreams: 0 / Made available in DSpace on 2014-12-19T16:34:27Z (GMT). No. of bitstreams: 0 / Tumores carcinoides (neoplasias bem diferenciadas) são tumores neuroendócrinos que podem surgir em diferentes locais anatômicos. Na população a prevalência dos tumores carcinoides é de aproximadamente 10 casos para um milhão de habitantes e sua incidência é maior na quinta e sexta década de vida. Este trabalho propõe um modelo cinético baseado na teoria da análise compartimental em humanos com tumores carcinoides que se submeterão ao tratamento com o radiofármaco [177Lu-DOTA0,Tyr3]Octreotato. Imagens cintilográficas dinâmicas planares, obtidas imediatamente à injeção de 370 MBq (10 mCi) do radiofármaco, foram obtidas com o tomógrafo SPECT (Single Photon Emission Computed Tomography). Por meio da seleção de regiões de interesse (ROI) os resultados foram digitalizados e aplicados ao modelo cinético aqui proposto. A primeira fase do estudo (atividade de 370 MBq) teve como objetivo conhecer os parâmetros cinéticos e subsequentemente, o paciente foi submetido ao protocolo de tratamento radioterápico, a critério médico, aos quatro ciclos de 7,4 GBq (200 mCi) do radiofármaco. Desta forma, foi possível estimar previamente as constantes cinéticas ki,j da biodistribuição do 177Lu-DOTATATO no corpo, sendo ki,j a fração de transferência do i-ésimo compartimento (tecido ou órgão) para o j-ésimo compartimento a partir das ROI demarcadoras dos órgãos de maior captação, a saber: fígado, rins, região vascularizada e tumores carcinoides. A partir das constantes cinéticas ki,j a estimativa de dose absorvida em 26 órgãos foi estimada pelo método MIRD. Os resultados dosimétricos foram compatíveis com outras metodologias descritas na literatura. Para um paciente adulto de 73,6 kg, em termos médios seus rins (sem os protetores renais) recebem a maior intensidade de dose (2,39 mGy/MBq) seguido do fígado (0,70 mGy/MBq). Observou-se que tumores com aproximadamente 100g recebem dose da ordem de 0,52 mGy/MBq independentemente da posição a que se encontram no corpo. Este achado se deve à predominância do dano devido às partículas beta quando comparado à radiação gama que possui pouco rendimento de emissão no processo de decaimento do 177Lu. Portanto, os parâmetros cinéticos que promovem a captação do 177Lu nas células são os principais responsáveis pela composição da dose no tumor e demais órgãos. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

In several human cancers, ErbB2 over-expression facilitates the formation of constitutively active homodimers resistant to internalization which results in progressive signal amplification from the receptor, conducive to cell survival, proliferation, or metastasis. Here we report on studies of the influence of ErbB2 over-expression on localization and signaling in polarized Caco-2 and MDCK cells, two established models to study molecular trafficking. In these cells, ErbB2 is not over-expressed and shares basolateral localization with ErbB3. Over-expression of ErbB2 by transient transfection resulted in partial separation of the receptors by relocalization of ErbB2, but not ErbB3, to the apical surface, as shown by biotinylation of the apical or basolateral surfaces. These results were confirmed by immunofluorescence and confocal microscopy. Polarity controls indicated that the relocalization of ErbB2 is not the result of depolarization of the cells. Biotinylation and confocal microscopy also showed that apical, but not basolateral ErbB2 is activated at tyrosine 1139. This phosphotyrosine binds adaptor protein Grb2, as confirmed by immunoprecipitation. However, we found that it does not initiate the canonical Grb2-Ras-Raf-Erk pathway. Instead, our data supports the activation of a survival pathway via Bcl-2. The effects of ErbB2 over-expression were abrogated by the humanized anti-ErbB2 monoclonal antibody Herceptin added only from the apical side. The ability of apical ErbB2 to initiate an altered downstream cascade suggests that subcellular localization of the receptor plays an important role in regulating ErbB2 signaling in polarized epithelia.

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